Tissue engineering may be defined as the art of reconstructing mammalian tissues, both structurally and functionally (Hunziker, 2002). In vitro tissue engineering generally includes the delivery of a polymeric scaffold that serves as an architectural support onto which cells may attach, proliferate, and synthesize new tissue to repair a wound or defect
An example of a tissue that is prone to damage by disease and trauma is the articular cartilage, one of several types of cartilage in the body, found at the articular surfaces of bones. Damage to cartilage may result from an inflammatory disease such as rheumatoid arthritis, from a degenerative process such as osteoarthritis or from trauma such as intraarticular fracture or following ligament injuries. Cartilage lesions are often associated with pain and reduced function and generally do not heal without medical intervention.
Current therapeutic strategies for repairing damaged cartilage include procedures which induce a spontaneous repair response and those which reconstruct the tissue in a structural and functional manner. The former, including surgical techniques such as abrasion artheroplasty, microfracture or subchondral micro-drilling, expose the subchondral region of bone thereby allowing the formation of a blood clot and infiltration of pluripotent stem cells to initiate the healing response. Often the induced tissue is not durable and the clinical improvements are short lived.
An alternative is transplantation of chondral or osteochondral tissue from either autologous or allogeneic sources. The rationale behind transplantation lies in the notion that the proliferative and tissue-differentiation activities of these cells would result in the formation of neocartilage. In fact, this technique shows high variability and limited clinical success.
Matrices useful for tissue regeneration and/or as biocompatible surfaces useful for tissue culture are well known in the art. These matrices may therefore be considered as substrates for cell growth either in vitro or in vivo. Suitable matrices for tissue growth and/or regeneration include both biodegradable and biostable entities. Among the many candidates that may serve as useful matrices claimed to support tissue growth or regeneration, are included gels, foams, sheets, and numerous porous particulate structures of different forms and shapes.
Porous materials formed from synthetic and/or naturally occurring biodegradable materials have been used in the past as wound dressings or implants. The porous material provides structural support and a framework for tissue in-growth while healing progresses. Preferably, the porous material is gradually absorbed as the tissue around the wound regenerates. Typical bioabsorbable materials for use in the fabrication of porous wound dressings or implants include both synthetic polymers and biopolymers such as structural proteins and polysaccharides. The biopolymers may be either selected or manipulated in ways that affect their physico-chemical properties. For example biopolymers may be cross linked either enzymatically, chemically or by other means, thereby providing greater or lesser degrees of flexibility or susceptibility to degradation.
Among the manifold natural polymers which have been disclosed to be useful for tissue engineering or culture, one can enumerate various constituents of the extracellular matrix including fibronectin, various types of collagen, and laminin, as well as keratin, fibrin and fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate and others.
Fibrin is a major plasma protein which participates in the blood coagulation process. The coagulation of blood is a complex process including the sequential interaction of a number of plasma proteins, in particular of fibrinogen (factor I); factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII and factor XIII.
Other plasma proteins such as Von Willebrand factor (vWF), albumin, immunoglobulin, coagulation factors, and complement components may also play a part in the formation of protein networks or clots in the blood.
Fibrin is known in the art as a tissue adhesive medical device and can be used in wound healing and tissue repair. Lyophilized plasma-derived protein concentrate (containing Factor XIII, fibronectin, and fibrinogen), in the presence of thrombin and calcium forms an injectable biological glue. U.S. Pat. No. 5,411,885 discloses a method of embedding and culturing tissue employing fibrin glue. U.S. Pat. No. 4,642,120 discloses the use of fibrinogen glue in combination with autologous mesenchymal or chondrocytic cells to promote repair of cartilage and bone defects. U.S. Pat. No. 5,260,420, discloses a method for preparation and usage of biological glue comprising fibrin for injection at the site of injury. U.S. Pat. No. 6,074,663 discloses a cross-linked fibrin sheet-like material for the prevention of adhesion formation.
U.S. Pat. No. 6,310,267 discloses a specific process for preparing a biodegradable flexible fibrin web for wound healing. The process necessitates dialyzing a fibrinogen solution with a solution containing chelators and forming the flexible web by the addition of a thrombin solution, freeze drying and Iyophilizing the web.
U.S. Pat. No. 5,972,385 discloses a cross-Linked collagen-polysaccharide matrix that is administered alone or in combination with other therapeutics, such as growth factors, for tissue repair. The invention also discloses the cross-linked collagen-polysaccharide matrix in combination with fibrin. The matrix preparation steps include freezing and lyophilization as well as adding fibrinogen and thrombin to form fibrin in said matrix.
A freeze-dried fibrin web for wound healing has been disclosed in U.S. Pat. No. 6,310,267. The preparation of the web, as disclosed, requires a single- or multistage dialysis of the fibrinogen solution. According to that disclosure, the single-stage or multistage dialysis of the fibrinogen solution crucially changes its composition and the concentration of salts and amino acids customarily contained in it are considerably reduced. The dialysis is carried out in an aqueous solution of a physiologically compatible inorganic salt such as NaCl and an organic complexing agent such as alkali metal salts of EDTA, of oxalic acid or of citric acid.
A fibrin sponge containing a blood clotting activator for hemostasis, tissue adhesion, wound healing and cell culture support is disclosed in WO 99/15209.
According to that disclosure, the restoration of moisture or water content following lyophilization is crucial for obtaining a soft, adaptable, highly, absorbent sponge.
U.S. Pat. Nos. 5,955,438 and 4,971,954 disclose collagen-based matrices cross-linked by sugars, useful for tissue regeneration. U.S. Pat. No.5,700,476 provides a bioabsorbable implant material containing pharmacologically active agents, suitable for use in wound repair. The method describes the mixture of two biopolymer components, freeze dried to form a heteromorphic sponge, that allows a phased release of a pharmacologically active agent.
U.S. Pat. No. 5,736,372 discloses a biodegradable synthetic polymeric fibrous matrix containing chondrocytes for in vivo production of a functional cartilaginous structure to be used in joint lining.
U.S. Pat. No. 5,948,429 discloses a method of preparing a biopolymer foam comprising forming a biopolymer solution, cross-linking said solution with ultraviolet radiation and subsequently freeze-drying to form a lattice.
U.S. Pat. No. 5,443,950 relates to a method for implantation of a variety of cell types growing on a three dimensional cell matrix which has been inoculated with stromal cells to form a three dimensional stromal matrix. Further disclosed in U.S. Pat. No. 5,842,477 is a method of in vivo cartilage repair by implanting a biocompatible three dimensional scaffold in combination with periosteal/perichondrial tissue and stromal cells, with or without bioactive agents, for the production of new cartilage at the site of implantation. The scaffold used is selected from a group consisting of biodegradable or non-biodegradable materials.
There is an unmet need for a treatment for tissue defects, including but not limited to those found in diseased or injured cartilage and bone. Nowhere in the background art is it taught or suggested that mechanical and physical parameters of freeze-dried matrices composed of plasma proteins can be controlled by use of auxiliary components or additives, which may be removed after the matrix is formed, in order to improve the biological properties of the matrix. The matrices of the invention are useful for cellular growth, as an implant per se and/or as a cell-bearing implant suitable for transplantation.